Adzuki and Mung Bean Sprouts Enriched with Probiotic Lactiplantibacillus plantarum 299v Improve Body Mass Gain and Antioxidant Status and Reduce the Undesirable Enzymatic Activity of Microbiota in Healthy Rats.

Introducing and establishing new food requires a detailed evaluation of its safety, nutritional value and functionality, thus the control and probiotic-rich adzuki and mung bean sprouts were studied in an in vivo rats model. However, the total feed intake did not differ significantly between the groups, the highest body weight gain and body weight change were recorded in the control AIN diet. At the same time, the addition of legume sprouts caused a reduction of these parameters (up to 25% in the variant with probiotic-rich adzuki bean sprouts). There was no significant effect on serum morphology, except white blood cells (ca. 20% reduction in the control sprout-supplemented diets). Serum and liver antiradical properties were significantly elevated by consuming mung bean sprouts (no effect of the probiotics). The faecal lactic acid bacteria were already increased by the control sprouts (a 2.8- and 2.1-fold increase for adzuki and mung bean sprouts, respectively). The probiotic-rich sprouts further improved this parameter. The diets enriched with mung bean sprouts significantly decreased the urease (by ca. 65%) and ß-glucuronidase activities (by ca. 30%). All the tested diets caused also a significant reduction of faecal tryptophanase activity (the effect was intensified by Lactiplantibacillus plantarum 299v). The functional components did not affect negatively the nutritional parameters and blood morphological characteristics. They improved also the antioxidant potential and significantly decreased the activities of colon cancer-related enzymes (urease and tryptophanase). The results confirmed that these new probiotic carriers may be a valuable, safe and functional element of a healthy diet.

Occurrence and genetic diversity of prophage sequences identified in the genomes of L. casei group bacteria.

It is widely believed that microorganisms belonging to L. casei group can have positive effects on the human body. Therefore, these bacteria are used in many industrial processes, including the production of dietary supplements and probiotic preparations. When using live microorganisms in technological processes, it is important to use those without phage sequences within their genomes that can ultimately lead to lysis of the bacteria. It has been shown that many prophages have a benign nature, meaning that they don't directly lead to lysis or inhibit microbial growth. Moreover, the presence of phage sequences in the genomes of these bacteria increases their genetic diversity, which may contribute to easier colonization of new ecological niches. In the 439 analyzed genomes of the L. casei group, 1509 sequences of prophage origin were detected. The average length of intact prophage sequences analyzed was just under 36 kb. GC content of tested sequences was similar for all analyzed species (44.6 ± 0.9%). Analyzing the protein coding sequences collectively, it was found that there was an average of 44 putative ORFs per genome, while the ORF density of all phage genomes varied from 0.5 to 2.1. The average nucleotide identity calculated on sequence alignments for analyzed sequences was 32.7%. Of the 56 L. casei strains used in the next part of the study, 32 did not show culture growth above the OD600 value of 0.5, even at a mitomycin C concentration of 0.25 µg/ml. Primers used for this study allowed for the detection of prophage sequences for over 90% of tested bacterial strains. Finally, prophages of selected strains were induced using mitomycin C, phage particles were isolated and then genomes of viruses obtained were sequenced and analyzed.

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level.

The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level-genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

A New Strategy for Effective Succinic Acid Production by Enterobacter sp. LU1 Using a Medium Based on Crude Glycerol and Whey Permeate.

The newly-isolated strain Enterobacter sp. LU1, which has previously been shown to be an effective producer of succinic acid on glycerol with the addition of lactose, was used for further intensive works aimed at improving the production parameters of the said process. The introduction of an initial stage of gentle culture aeration allowed almost 47 g/L of succinic acid to be obtained after 168 h of incubation, which is almost two times faster than the time previously taken to obtain this amount. Furthermore, the replacement of glycerol with crude glycerin and the replacement of lactose with whey permeate allowed the final concentration of succinic acid to be increased to 54 g/L. Considering the high content of yeast extract (YE) in the culture medium, tests were also performed with a reduced YE content via its partial substitution with urea. Although this substitution led to a deterioration of the kinetic parameters of the production process, using the fed-batch strategy, it allowed a succinic acid concentration of 69 g/L to be obtained in the culture medium, the highest concentration ever achieved using this process. Furthermore, the use of microaerophilic conditions meant that the addition of lactose to the medium was not required, with 37 g/L of succinic acid being produced on crude glycerol alone.

Genomic and Proteomic Characterization of Bacteriophage BH1 Spontaneously Released from Probiotic Lactobacillus rhamnosus Pen.

Lactobacillus rhamnosus Pen is a human endogenous strain used for the production of probiotic formula, which is effective in the prevention of antibiotic-associated diarrhoea. Our study showed that this probiotic strain releases bacteriophage BH1 without the addition of any inducing agent. Our research revealed that phage BH1 has a circular genome with a length of 40721 nt and a GC content of 44.8%. The genome of phage BH1 possesses 57 open reading frames which could be divided into functional modules associated with DNA packaging, morphogenesis, lysis, integration, genetic switch, and replication. In spite of similarity in morphology and genomic organization, comparative analysis revealed substantial genetic diversity and mosaic genomic architecture among phages described for the Lactobacillus casei group. Additionally, qPCR and ddPCR analysis confirmed earlier microscopic observations indicating that L. rhamnosus Pen liberates bacteriophage particles during growth. This occurs spontaneously, and is not a result of external inducing factors. For samples collected after 4 and 24 h of L. rhamnosus Pen culture, the number of attB and attP copies increased 2.5 and 12 times, respectively. This phenomenon, by introducing resistance to other phages or enhancing the biofilm-forming capabilities, may increase the survivability of microorganisms in their natural ecological niche. Conversely, spontaneous phage induction may be an important virulence factor for bacteria, posing a potential threat for the human host.

Complete genome sequence of Lactobacillus rhamnosus Pen, a probiotic component of a medicine used in prevention of antibiotic-associated diarrhoea in children.

BACKGROUND: Lactobacillus rhamnosus Pen is a human endogenous strain with well-documented health promoting properties that is used for production of probiotics. It has a long safety history of application, and its effectiveness in the prevention of antibiotic-associated diarrhoea has also been confirmed in clinical trials. RESULTS: Here we present the complete genome sequence of L. rhamnosus Pen, which consists of a circular 2,884,4966-bp chromosome with a GC content of 46.8%. Within 2907 open reading frames (ORFs), genes involved with probiotic properties were identified. A CRISPR locus, consisting of a 1092-nt region with 16 spacers, was also detected. Finally, an intact prophage of ~ 40.7 kb, 57 ORFs, GC content 44.8% was identified. CONCLUSIONS: Genomic analysis confirmed the probiotic properties of L. rhamnosus Pen and may indicate new biotechnological applications of this industrially important strain.

Mechanical thrombectomy in acute stroke - Five years of experience in Poland.

OBJECTIVES: Mechanical thrombectomy (MT) is not reimbursed by the Polish public health system. We present a description of 5 years of experience with MT in acute stroke in Comprehensive Stroke Centers (CSCs) in Poland. METHODS AND RESULTS: We retrospectively analyzed the results of a structured questionnaire from 23 out of 25 identified CSCs and 22 data sets that include 61 clinical, radiological and outcome measures. RESULTS: Most of the CSCs (74%) were founded at University Hospitals and most (65.2%) work round the clock. In 78.3% of them, the working teams are composed of neurologists and neuro-radiologists. All CSCs perform CT and angio-CT before MT. In total 586 patients were subjected to MT and data from 531 of them were analyzed. Mean time laps from stroke onset to groin puncture was 250±99min. 90.3% of the studied patients had MT within 6h from stroke onset; 59.3% of them were treated with IV rt-PA prior to MT; 15.1% had IA rt-PA during MT and 4.7% - emergent stenting of a large vessel. M1 of MCA was occluded in 47.8% of cases. The Solitaire device was used in 53% of cases. Successful recanalization (TICI2b-TICI3) was achieved in 64.6% of cases and 53.4% of patients did not experience hemorrhagic transformation. Clinical improvement on discharge was noticed in 53.7% of cases, futile recanalization - in 30.7%, mRS of 0-2 - in 31.4% and mRS of 6 in 22% of cases. CONCLUSION: Our results can help harmonize standards for MT in Poland according to international guidelines.

Media optimization for economic succinic acid production by Enterobacter sp. LU1.

Enterobacter sp. LU1 could efficiently convert glycerol to succinic acid under anaerobic conditions after the addition of lactose. In this study, media constituents affecting both Enterobacter sp. LU1 biomass and succinic acid production were investigated employing response surface methodology (RSM) with central composite design. Statistical methods led to the development of an efficient and inexpensive microbiological media based on crude glycerol, whey permeate as carbon sources and urea as a nitrogen source. The optimized production of bacterial biomass in aerobic conditions was predicted and the interactive effects between crude glycerol, urea and magnesium sulfate were investigated. As a result, a model for predicting the concentration of bacterial biocatalyst biomass was developed with crude glycerol as a sole carbon source. In addition, it was observed that the interactive effect between crude glycerol and urea was statistically significant. Response surface methodology was also employed to develop the model for predicting the concentration of succinic acid produced. Validity of the model was confirmed during verification experiments wherein actual results differed from predicted values by 0.77%. The applied statistical methods proved the feasibility for anaerobic succinic acid production on crude glycerol without expensive yeast extract addition. In conclusion, the RSM method can provide valuable information for succinic acid scale-up fermentation using Enterobacter sp. LU1.

Enterobacter sp. LU1 as a novel succinic acid producer - co-utilization of glycerol and lactose.

Succinic acid is an important C4-building chemical platform for many applications. A novel succinic acid-producing bacterial strain was isolated from goat rumen. Phylogenetic analysis based on the 16S rRNA sequence and physiological analysis indicated that the strain belongs to the genus Enterobacter. This is the first report of a wild bacterial strain from the genus Enterobacter that is capable of efficient succinic acid production. Co-fermentation of glycerol and lactose significantly improved glycerol utilization under anaerobic conditions, debottlenecking the utilization pathway of this valuable biodiesel waste product. Succinic acid production reached 35 g l-1 when Enterobacter sp. LU1 was cultured in medium containing 50 g l-1 of glycerol and 25 g l-1 of lactose as carbon sources.

Comparison of various molecular methods for rapid differentiation of intestinal bifidobacteria at the species, subspecies and strain level.

BACKGROUND: Members of the genus Bifidobacterium are anaerobic Gram-positive Actinobacteria, which are natural inhabitants of human and animal gastrointestinal tract. Certain bifidobacteria are frequently used as food additives and probiotic pharmaceuticals, because of their various health-promoting properties. Due to the enormous demand on probiotic bacteria, manufacture of high-quality products containing living microorganisms requires rapid and accurate identification of specific bacteria. Additionally, isolation of new industrial bacteria from various environments may lead to multiple isolations of the same strain, therefore, it is important to apply rapid, low-cost and effective procedures differentiating bifidobacteria at the intra-species level. The identification of new isolates using microbiological and biochemical methods is difficult, but the accurate characterization of isolated strains may be achieved using a polyphasic approach that includes classical phenotypic methods and molecular procedures. However, some of these procedures are time-consuming and cumbersome, particularly when a large group of new isolates is typed, while some other approaches may have too low discriminatory power to distinguish closely related isolates obtained from similar sources. RESULTS: This work presents the evaluation of the discriminatory power of four molecular methods (ARDRA, RAPD-PCR, rep-PCR and SDS-PAGE fingerprinting) that are extensively used for fast differentiation of bifidobacteria up to the strain level. Our experiments included 17 reference strains and showed that in comparison to ARDRA, genotypic fingerprinting procedures (RAPD and rep-PCR) seemed to be less reproducible, however, they allowed to differentiate the tested microorganisms even at the intra-species level. In general, RAPD and rep-PCR have similar discriminatory power, though, in some instances more than one oligonucleotide needs to be used in random amplified polymorphic DNA analysis. Moreover, the results also demonstrated a high discriminatory power of SDS-PAGE fingerprinting of whole-cell proteins. On the other hand, the protein profiles obtained were rather complex, and therefore, difficult to analyze. CONCLUSIONS: Among the tested procedures, rep-PCR proved to be the most effective and reliable method allowing rapid differentiation of Bifidobacterium strains. Additionally, the use of the BOXA1R primer in the differentiation of 21 Bifidobacterium strains, newly isolated from infant feces, demonstrated slightly better discriminatory power in comparison to PCR reactions with the (GTG)5 oligonucleotide. Thus, BOX-PCR turned out to be the most appropriate and convenient molecular technique in differentiating Bifidobacterium strains at all taxonomic levels.

A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

Genetic diversity of bile salt hydrolases among human intestinal bifidobacteria.

This study analyzes the application of degenerative primers for the screening of bile salt hydrolase-encoding genes (bsh) in various intestinal bifidobacteria. In the first stage, the design and evaluation of the universal PCR primers for amplifying the partial coding sequence of bile salt hydrolase in bifidobacteria were performed. The amplified bsh gene fragments were sequenced and the obtained sequences were compared to the bsh genes present in GenBank. The determined results showed the utility of the designed PCR primers for the amplification of partial gene encoding bile salt hydrolase in different intestinal bifidobacteria. Moreover, sequence analysis revealed that bile salt hydrolase-encoding genes may be used as valuable molecular markers for phylogenetic studies and identification of even closely related members of the genus Bifidobacterium.

Spontaneous release of bacteriophage particles by Lactobacillus rhamnosus pen.

The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

Molecular characterization of bile salt hydrolase from Bifidobacterium animalis subsp. lactis Bi30.

The present work describes the identification, purification, and characterization of bile salt hydrolase (BSH) from Bifidobacterium animalis subsp. lactis. The enzyme was purified to electrophoretic homogeneity by hydrophobic chromatography, ion-exchange chromatography and ultrafiltration. SDS-PAGE analysis of putative BSH and gel filtration revealed that the analyzed protein is presumably a tetramer composed of four monomers each of about 35 kDa. The purified enzyme was analyzed by liquid chromatography coupled to LTQ FT ICR mass spectrometry and unambiguously identified as a bile salt hydrolase from B. animalis. The isoelectric point of the studied protein was estimated to be around pH 4.9. The pH optimum of the purified BSH is between 4.7 to 6.5, and the temperature optimum is around 50 degrees C. The BSH of B. animalis could deconjugate all tested bile salts, with clear preference for glycine-conjugated bile salts over taurine-conjugated forms. Genetic analysis of the bsh showed high similarity to the previously sequenced bsh gene from B. animalis and confirmed the usefulness of bile salt hydrolase as a genetic marker for B. animalis identification.

LC-MS/MS analysis of surface layer proteins as a useful method for the identification of lactobacilli from the Lactobacillus acidophilus group.

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/ MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

[Usefulness of the antisense and triplex anti-IGF-1 techniques for postoperative cellular gene therapy of malignant gliomas expressing IGF-1]. / Pooperacyjna terapia genokomórkowa z ekspresja IGF-1 glejaka wielopostaciowego przy zastosowaniu techniki antysensu i tripleksu anty-IGF-1.

BACKGROUND AND PURPOSE: The aim of the study was to estimate the usefulness of the antineoplastic vaccination in treatment of malignant brain tumors. According to medical knowledge there is no cure for this kind of tumors. MATERIAL AND METHODS: Between 2001 and 2005, ten patients suffering from malignant glial tumors were treated. There were 5 male and 5 female individuals, aged from 17 to 76 (mean age: 40.8 years). The histopathological examination showed 4 cases of glioblastoma and 6 cases of anaplastic astrocytoma. Initially, patients were operated on with dissection of 1 cm(3) of the most representative part of tumor. The neoplasm cells were cultured, transfected with episomal pMT EP vector (expressing alternatively oligonucleotide sequence forming triple helix with IGF-I gene or antisense against IGF-1 mRNA), re-cultured, irradiated and resuspended in medium to prepare antineoplastic vaccine. The patients were vaccinated subcutaneously. We examined peripheral blood lymphocyte subsets to assess the immunological response of the patients. RESULTS: We observed prolongation of the survival time to 21.7 months compared to 9-11 months observed in literature. The patients were additionally treated oncologically with radiotherapy and chemotherapy (temozolomide) according to the reasonable indications. Therefore, the comprehensive assessment of the genotherapy as the supplemental monotherapy was impossible. CONCLUSIONS: The method of treatment used in this study prolongs the survival time of patients with high-grade gliomas of the central nervous system. This gene therapy needs further investigations as a method of oncological monotherapy of brain malignant gliomas.